quantification of sterol and triterpenol biomarkers in sediments of

Research ArticleQuantification of Sterol and Triterpenol Biomarkers inSediments of the Cananeacuteia-Iguape Estuarine-Lagoonal System(Brazil) by UHPLC-MSMS

Giovana Anceski Bataglion12 Hector Henrique Ferreira Koolen3

Rolf Roland Weber4 and Marcos Nogueira Eberlin2

1Department of Chemistry Federal University of Amazonas (UFAM) 69077-000 Manaus AM Brazil2ThoMSon Mass Spectrometry Laboratory Institute of Chemistry University of Campinas (UNICAMP)13083-970 Campinas SP Brazil3DeMpSter Mass Spectrometry Group Amazonas State University (UEA) 69050-010 Manaus AM Brazil4Marine Organic Chemistry Laboratory Oceanography Institute University of Sao Paulo (USP) 05508-120 Sao Paulo SP Brazil

Correspondence should be addressed to Giovana Anceski Bataglion giovanabataglionufamedubr andHector Henrique Ferreira Koolen hectorkoolengmailcom

Received 31 December 2015 Accepted 6 March 2016

Academic Editor Josep Esteve-Romero

Copyright copy 2016 Giovana Anceski Bataglion et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Sterols and triterpenols present in sedimentary cores from 12 stations along the Cananeia-Iguape estuarine-lagoonal system wereinvestigated by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MSMS) Ten sterols andthree triterpenols were identified and quantified indicating both natural and anthropogenic sources The relative distributionsof sterol and triterpenol showed that the study area is submitted to organic matter (OM) from the Ribeira de Iguape River seawatersurrounding vegetation and plankton production The contribution of these sources depends on the region of the estuarine-lagoonal system and the depth of sediment Regarding anthropogenic sources only the samples submitted to freshwater flowfrom the Ribeira de Iguape River presented concentration of coprostanol higher than the threshold value and diagnostic ratioscoprostanol(coprostanol + cholestanol) and coprostanolcholesterol that indicate moderate contamination by domestic sewagein that area of the estuarine-lagoonal system Therefore the approach used herein identified the OM sources and its transportalong the Cananeia-Iguape estuarine-lagoonal system (Brazil) which is a complex of lagoonal channels located in a United NationsEducational Scientific and Cultural Organization (UNESCO) Biosphere Reserve

1 Introduction

Sterols also known as alcohol steroids are hydrophobiccompounds that occur naturally in most of the living forms[1] This class of compounds can be easily incorporated intosediments where they remain well preserved representinggood tracers of sources changes and preservation of organicmatter (OM) [2] Sterols have been used to assess the OMorigin in several estuarine systems around the world becauseof their recalcitrance and specific-source which allowsclassifying the OM input from different sources includingterrestrial plants [3] phytoplankton [4] bacteria [5] and

heterotrophic eukaryote and benthic algae [6] Furthermorebesides natural OM sources sterols have been applied inenvironmental studies to track human activities in coastalareas [7 8]

Phytosterols sterols C28

and C29 comprise the major

sterol type found in estuarine sediments [9] Campesterol120573-sitosterol stigmasterol and stigmastanol are plant sterolsthus they are used as geochemical biomarkers to assess theplant-derivedOMinput to aquatic systems [9 10] Algae oftendisplay predominance of C

27sterols (mainly cholesterol)

with compositions different from those observed for vascularplants [11] In addition brassicasterol is frequently found in

Hindawi Publishing CorporationInternational Journal of Analytical ChemistryVolume 2016 Article ID 8361375 8 pageshttpdxdoiorg10115520168361375

2 International Journal of Analytical Chemistry

several algal classes whereas cholestanol often occurs in thedinoflagellates class [11] Other sterols such as dinosterol andergosterol are exclusive biomarkers of OM from diatoms andfungi respectively [12]

There are also important sterol biomarkers for anthro-pogenic OM which are known as fecal sterols (egcoprostanol and epicoprostanol) and indicate contaminationfrom domestic sewage [13] Some authors point that absoluteconcentration of coprostanol should not be used as a uniqueand conclusive parameter to assess sewage contaminationbecause of the possibility of in situ production To solve thisissue and to evaluate the degree of contamination by domesticsewage input some ratios such as coprostanol(coprostanol+ cholestanol) and coprostanolcholesterol have been pro-posed [14] Higher plants especially angiosperms displaytriterpenols as typical and exclusive biomarkers Triterpenolssuch as lupeol and 120572- and 120573-amyrin are the most commontriterpenols found in sediments of riverine and estuarineenvironments [15]

Estuarine systems are essential compartments involvedin the OM recycling and global carbon cycle [16] Under-standing the flows of OM frommarine and rivers to estuariesprovides information about its cycle in both water columnand sediments [17] Once OM in these systems constitutesa complex mixture of compounds from different sources areliable technique is required to overcome this complexity[18] Sterols in sediment samples are generally analyzed bygas chromatography-mass spectrometry (GC-MS) whichrequires a cleanup step using alumina and silica columnswhere sterols are separated from other classes of compoundsThe fraction containing sterols is usually derivatized toincrease their volatility and decrease the limits of detec-tion (LOD) [19] In this sense liquid chromatography (LC)as a separation technique and tandem mass spectrometry(MSMS) as a detection method are combined to over-come the inherent challenges of OM complexity [15] Somemethods employing analytical platforms based on liquidchromatograph tandem mass spectrometry (LC-MSMS) forthe analysis of selected sterols have been described over thelast years for a range of matrices [20 21] and more recentlyfor sediments [15]

Information on sources transport and deposition of OMis scarce for the Cananeia-Iguape estuarine-lagoonal systemwhich is an important Brazilian ecosystem from the WorldHeritage List under natural criteria (UNESCO) due to exten-sive areas of mangrove and Atlantic forest Consequentlythis study aimed to determine the sterol and triterpenolcomposition and assess the OM origin in sedimentary coresalong the study area Based on that an initial insight intothe geochemistry and flow of OM in this estuarine-lagoonalenvironment was achieved

2 Experimental

21 Study Area and Sampling The Cananeia-Iguape estua-rine-lagoonal system (Figure 1) is located in the SoutheastRegion of Brazil (Sao Paulo state) The opening of a 4 kmlong artificial channel linking the Ribeira de Iguape River tothe Cananeia-Iguape estuarine-lagoonal system in the XIX

Icapara outfall

Cananeacuteia outfall

Ribeira de Iguape River

Atlantic Ocean

S

N

W E

S1S2

S3

S4

S5S6S7

S8

S9S10

S11 S12

5 10 200(km)

Figure 1 Map of study area showing the 12 sampling stationsalong the Cananeia-Iguape estuarine-lagoonal system located inSao Paulo state (Southeast Brazil) Content may not reflect NationalGeographicrsquos current map policy Sources National GeographicEsri DeLorme HERE UNEP-WCMC USGS NASA ESA METINRCAN GEBCO NOAA and Increment P Corp

century caused an enormous impact in this environmentAfter its opening the channel eroded laterally reaching awidth and depth of approximately 250 and 7m respectivelyThus the channel was renamed to Valo Grande (ldquoBig Scourrdquo)in reference to the dimensions that it acquired As a result ofthis erosion environmental changes in the Cananeia-Iguapeestuarine-lagoonal system were intensified since about 60of the main flow of the Ribeira de Iguape River was trans-ferred to the system [22] The affected area is an importantecosystem from the World Heritage List located betweentwo cities (Cananeia and Iguape) with a protected greenarea consisting of typical vegetation of salt marshes Atlanticforest and mangroves [22]

A total of 60 samples from 12 collection sites (0ndash10 cmdepth) were collected at the Cananeia-Iguape estuarine-lagoonal system Sampling sites with their geographicallocations and lithological characteristics are shown in Table 1Sedimentary core samples were collected using a RossfelderVT-1 vibracorer and stored at 4∘C during transportation tothe laboratory Prior to the laboratory work all sedimentsamples were freeze-dried pulverized in a mortar and sievedwith a stainless steel sieve (lt250120583m) and stored at 4∘C beforeanalysis

22 Chemicals and Reagents (3120573)-Cholest-5-en-3-ol (cho-lesterol ge99 purity) (31205735120572)-cholestan-3-ol (cholestanolgt95 purity) (31205735120573)-cholestan-3-ol (coprostanol gt95purity) (31205725120573)-cholestan-3-ol (epicoprostanol gt95 pu-rity) (312057322E)-ergosta-5722-trien-3-ol (ergosterol gt95purity) (312057322E)-ergosta-522-dien-3-ol (brassicasterolgt95 purity) (312057324R)-ergost-5-en-3-ol (campesterol gt65purity) (3120573)-stigmast-5-en-3-ol (120573-sitosterol gt98 purity)(312057322E)-stigmasta-522-dien-3-ol (stigmasterol gt95 puri-ty) (31205735120572)-stigmastan-3-ol (stigmastanol gt95 purity)and (3120573)-lup-20(29)-en-3-ol (lupeol ge94 purity) were

International Journal of Analytical Chemistry 3

Table 1 Geographical locations and lithological characteristics for the 12 sampling sites in the Cananeia-Iguape estuarine-lagoonal system

Station Longitude (W) Latitude (S) Lithological characteristics1 47∘33101584077510158401015840 24∘42101584085510158401015840 Similar proportion of sand silt and clay2 47∘33101584071110158401015840 24∘43101584018010158401015840 Predominance of silt and clay3 47∘43101584048010158401015840 24∘42101584012710158401015840 Predominance of silt and clay4 47∘51101584094310158401015840 24∘42101584002210158401015840 Predominance of sand (gt80)5 47∘37101584009310158401015840 24∘45101584008010158401015840 Predominance of sand (gt70)6 47∘45101584009310158401015840 24∘49101584008010158401015840 Predominance of sand (gt80)7 47∘51101584044910158401015840 24∘54101584001010158401015840 Predominance of sand (gt90)8 47∘52101584007110158401015840 24∘57101584072310158401015840 100 of sand in the first 4 cm9 47∘55101584016810158401015840 25∘01101584002010158401015840 Predominance of sand (gt70)10 48∘01101584021410158401015840 25∘01101584001710158401015840 Similar proportion of sand silt and clay11 47∘58101584004710158401015840 25∘04101584008810158401015840 Predominance of clay (50)12 47∘55101584069310158401015840 25∘04101584027810158401015840 Predominance of sand (gt60)

purchased from Sigma-Aldrich (St Louis MO USA) The120572-amyrin ((3120573)-urs-12-en-3-ol) and 120573-amyrin ((3120573)-olean-12-en-3-ol) were isolated [23] and used only to determinetheir retention times and MRM transitions Based on thestructural similarities between all of the sterols and theirretention times cholesterol-119889

6(gt98 purity) was used

as internal standard (IS) for quantification of all of thecompounds HPLC-grade ethanol methanol n-hexane anddichloromethane were obtained from JTBaker (MexicoCity DF Mexico) and the water was purified by a Milli-Qgradient system (Millipore Milford MA USA)

23 Sediment Extractions The freeze-dried sediment sam-ples were macerated and ca 20 g was spiked with IS solutionto obtain a final concentration of 500 ngmL and they werethen extracted with 50mL of n-hexane and dichloromethane(1 1 vv) The extraction was performed using a microwavesystem MarsX (CEM Mathews NC USA) operating at1600W with pressure and temperature gradients reaching200 psi and 85∘C in 5min respectively The system was keptisothermal during 15min and then it returned to the roompressure and temperature conditions The organic extractswere evaporated to 1mL and an aliquot of 50 120583L was dilutedto exactly 1000 120583L of methanol

24 Instrumentation and Chromatographic Conditions Anal-ysis of the sterols and triterpenols in the sedimentaryOM extracts was conducted using an ultra-high perfor-mance liquid chromatography tandem mass spectrometry(UHPLC-MSMS) system model LCMS-8040 (ShimadzuKyoto Japan) The mass spectrometer system consists of atriple quadrupole equipped with an atmospheric pressurechemical ionization (APCI) probe The chromatographicseparation was performed on a Shimpack XR-ODS 22 120583m20mm id and 150mm column (Shimadzu Kyoto Japan)The gradient elution at 30∘C was as follows 0ndash2min (90B) 2ndash8min (100 B) and 8-9min (90 B) at a flow rateof 06mLmin Solvent A was water and solvent B wasmethanol

APCI source was operated in the positive ion modewith the parameters as the following corona current 40 120583A

heat block temperature 300∘C desolvation line temperature250∘CTheprecursor ionswere determined in full scan exper-iments and the product ions were chosen by MSMS experi-ments and then the analyses were conducted by multiple ionmonitoring (MRM) using two transitions for each standardcompound (Table S1 in Supplementary Material availableonline at httpdxdoiorg10115520168361375) dwell timeof 20ms and argon gas (99996 purity fromWhiteMartinsSao Paulo SP Brazil) pressure of 224 kPa and no dryinggas flow was used Data were acquired and processed by theLabSolutions software version 553 SP2 (Shimadzu KyotoJapan)

3 Results and Discussion

31 Quantitative Determination of Sterol and TriterpenolBiomarkers by UHPLC-MSMS The UHPLC-MSMSmethod using APCI provided high sensitivity and selectivityand required no derivatization of sterols and triterpenolsThemost abundant signal in the full scan spectra correspondedto the neutral loss of water from the protonated sterol whichis consistent with previous studies [20 21] and was chosen asprecursor ion The two most abundant signals in the MSMSspectra were chosen as the product ions The UHPLC-MSMS method for determination of sterols and triterpenolswas previously validated in relation to linearity intradayand interday assays of accuracy and precision and recovery[15] A representative UHPLC-MSMS chromatogram forthe analytes in a standard solution mixture is shown inFigure 2

Figure 2 shows the total ion chromatograms (TIC) forthe compounds which are the sum of the quantification andconfirmation transitions Peaks 2 6 and 9 were properlyseparated corresponding to the sequential elution of theisomers epicoprostanol coprostanol and cholestanol Peak3 corresponds to the elution of lupeol but its transitionsalso correspond to 120572- and 120573-amyrin which present differentretention times and were quantified using the calibrationcurve for lupeol More details about the validation of theUHPLC-MSMS method are described by Bataglion et al[15]


Table 2 Total concentrations of sterols and triterpenols (ngg of dry weight) in sedimentary core samples from the Cananeia-Iguapeestuarine-lagoonal system (119899 = 3)

Stations 0ndash2 cm 2ndash4 cm 4ndash6 cm 6ndash8 cm 8ndash10 cm1 1337 plusmn 67 1076 plusmn 140 680 plusmn 75 873 plusmn 87 1265 plusmn 632 1335 plusmn 93 1315 plusmn 145 283 plusmn 25 585 plusmn 53 420 plusmn 343 1480 plusmn 74 1157 plusmn 116 679 plusmn 54 433 plusmn 30 466 plusmn 514 1023 plusmn 92 804 plusmn 88 420 plusmn 34 517 plusmn 52 669 plusmn 875 1796 plusmn 215 751 plusmn 53 1187 plusmn 117 309 plusmn 31 582 plusmn 356 2180 plusmn 239 899 plusmn 54 2429 plusmn 194 1556 plusmn 124 805 plusmn 807 2526 plusmn 152 1162 plusmn 93 916 plusmn 64 586 plusmn 41 378 plusmn 308 140 plusmn 8 293 plusmn 23 1912 plusmn 134 2272 plusmn 159 2784 plusmn 1679 1377 plusmn 96 946 plusmn 57 677 plusmn 41 894 plusmn 54 1126 plusmn 7910 1870 plusmn 205 1860 plusmn 93 1762 plusmn 141 2624 plusmn 210 2298 plusmn 16111 1772 plusmn 195 1678 plusmn 134 1597 plusmn 143 1879 plusmn 169 1975 plusmn 15812 911 plusmn 73 831 plusmn 58 430 plusmn 26 520 plusmn 36 795 plusmn 87

12

3

45 6

7

8910

110

250000500000750000

100000012500001500000

Inte

nsity

10 20 30 40 50 60 70 80 9000Retention time (min)

Figure 2 Representative UHPLC-MSMS chromatogram for theanalytes in a standard solution mixture (20 ngmL) Ergosterol (1)epicoprostanol (2) lupeol (3) brassicasterol (4) cholesterol (5)coprostanol (6) campesterol (7) 120573-sitosterol (8) cholestanol (9)stigmasterol (10) and stigmastanol (11)

32 Total Content of Sterol and Triterpenol BiomarkersThe validated UHPLC-MSMS method was then applied toquantify sterols and triterpenols in 12 sedimentary cores fromthe Cananeia-Iguape estuarine-lagoonal system Table 2 liststhe total concentrations of the sterols and triterpenols inthe sedimentary core samples collected along the Cananeia-Iguape estuarine-lagoonal system

The total concentrations of sterols and triterpenols alongthe sedimentary core samples cover a wide range from 140to 2784 ngg dry weight This significant variability may beassociated with both lithological features of each sedimentarycore and temporal changes in primary productivity or inputof allochthonous OM The total concentrations of sterolsand triterpenols showed similar tendency along sedimentarycores 1 2 and 3 which were collected closer to the Ribeira deIguape River These samples are characterized by a mixtureof silt clay and fine sand rich in OM which is due to theinput ofmaterials from the Ribeira de Iguape River Althoughsamples 4 and 5 are dominated by sand particles the totalconcentrations of sterols and triterpenols were similar tothose observed for the samples near to the Ribeira deIguape River Located in the middle of the estuarine-lagoonalsystem samples 6 and 7 are predominantly sandy and werethe ones with higher total concentrations of sterols and

triterpenols at 0ndash2 cmdepth Also located in themiddle of theestuarine-lagoonal system sample 8 is completely constitutedby sand particles in the first 4 cm where the amount of fineparticles (silt and clay) increases These lithological featurescan be associated with the smallest and the highest totalconcentration at 0ndash2 cm and 8ndash10 cm respectively Locatedin the southern sector samples 9 10 11 and 12 present amixture of silt clay and thin sand with samples 9 and 12being the most sandy as a consequence of their proximityto the sea Sample 10 showed the lower variability alongthe sedimentary core which can be related to its locationnot affected by the inflow of seawater to the estuarine-lagoon system In this group of samples 9 and 12 werethe ones with the lowest total concentrations of sterols andtriterpenols along the sedimentary cores because of both thehigher proportion of sand particles and influence of seawaterinflow

33 Profile of Sterol and Triterpenol Biomarkers Figure 3shows that the relative distribution of sterol and triterpenolbiomarkers along the Cananeia-Iguape estuarine-lagoonalsystem presents a typical profile for each specific regionSedimentary cores collected at stations 1 2 and 3 (0ndash2 cm) present similar relative distributions of sterols andtriterpenols with cholesterol and 120573-sitosterol as the majorcompounds These sterols are representative of different OMsources 120573-sitosterol is one of the major sterols found inhigher plants whereas cholesterol is generally attributed toboth zooplankton and phytoplankton (autochthonous OM)[24ndash26] Although 120573-sitosterol is used as a biomarker ofterrestrial OM in several geochemical studies some authorsalso suggest the possibility of its origin fromphytoplankton oreven aquatic macrophytes [24 27] The relative contributionof these sources depends on both the studied environmentand influence of such different OM sources On the otherhand triterpenols such as 120572-amyrin 120573-amyrin and lupeolare considered highly specific geochemical biomarkers forhigher plants [24 28] The three stations located near tothe Ribeira de Iguape River are submitted to a high inputof freshwater containing both terrestrial OM and nutrients


0

10

20

Rela

tive (

)

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

Relat

ive (

)

0

10

20

Rela

tive (

)

S1S2

S3

S4 S5 S6

S7 S8 S9

S10 S11 S12

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

40

50

60

Rela

tive (

)

2 3 4 5 6 7 8 9 10 11 12 131Compound

1 3 4 5 6 7 8 9 10 11 12 132Compound

1 3 4 5 6 7 8 9 10 11 12 132Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

40

Rela

tive (

)

0

10

20

30

40

50

Rela

tive (

)

0

10

20

30

40

Rela

tive (

)

010203040506070

Rela

tive (

)

0

10

20

30

Rela

tive (

)

0

10

20

30

Relat

ive (

)

0

10

20

30

40

50

Rela

tive (

)

0

10

20

30

40

50

Rela

tive (

)

Figure 3 Relative distribution of sterols and triterpenols in sedimentary core samples at 0ndash2 cm from Cananeia-Iguape estuarine-lagoonalsystem as revealed by UHPLC-MSMS (1) Ergosterol (2) epicoprostanol (3) coprostanol (4) cholestanol (5) cholesterol (6) campesterol(7) stigmasterol (8) 120573-sitosterol (9) stigmastanol (10) brassicasterol (11) lupeol (12) 120573-amyrin and (13) 120572-amyrin

Therefore stations 1 2 and 3 are under influence of OM fromhigher plants aquatic plants and also plankton productionwhich justify the variety of compounds in the profiles ofFigure 3

The relative distribution of sterol and triterpenolbiomarkers for the sedimentary cores collected at stations4 and 5 (0ndash2 cm) was dominated by cholesterol indicatingaccentuated input of OM from aquatic organisms to thesestations This region is under influence of freshwater from

the Ribeira de Iguape River but also of seawater that enters inthe estuarine-lagoonal system by two outfalls (Cananeia andIcapara Figure 1) Thus the higher proportion of cholesterolin stations 4 and 5 is a result of the contribution of OMpresent in the seawater which commonly presents it as themajor sterol The sedimentary cores collected at stations 6 7and 8 also present high contribution of cholesterol howevera significant proportion of 120573-amyrin can be observed mainlyfor stations 6 and 7


For all of the samples collected near to Cananeia Islandstations 9 10 11 and 12 (0ndash2 cm) the triterpenol 120573-amyrinwas observed as the main compound indicating predomi-nance ofOM fromhigher plants Indeed the Cananeia regionis characterized by intense surrounding vegetation mainlymangrove forest around the channels [22] High contribu-tion of the triterpenol 120573-amyrin was already observed forestuaries where themangrove vegetationRhizophoramangle(red mangrove) and Avicennia germinans (black mangrove)is dominant [29]

The relative distributions of sterol and triterpenolbiomarkers for deeper samples (4ndash10 cm) of stations 1 2 and3 present similar profiles to those shown for samples 0ndash2 cmbut with a slight increase of 120573-sitosterol over cholesterolThisresult may be associated with an increase of the planktonicproduction as a result of the high input of freshwater richin nutrients from the Ribeira de Iguape River Althoughthe relative contribution of 120573-sitosterol was also higher fordeeper samples of sedimentary core collected at station 4cholesterol was always their major sterolThis slighter changeindicates that station 4 is weakly submitted to freshwaterfrom the Ribeira de Iguape River An opposite situationoccurs at station 5 where the relative distributions of steroland triterpenol biomarkers for deeper samples (4ndash10 cm)become more similar to those obtained for stations near tothe Ribeira de Iguape River For stations 6 7 and 8 a decreaseof the relative contribution of cholesterol is observed fordeeper samples and the sterol and triterpenol distributionsbecome more similar to those obtained for sedimentary coresamples collected at Cananeia Island For these ones 120573-amyrin and 120573-sitosterol were the compounds with higherrelative proportion along all the sedimentary cores

Based on the relative distributions of sterol and triter-penol biomarkers sediments from the Cananeia-Iguape estu-arine system are submitted to OM from the Ribeira de IguapeRiver seawater surrounding vegetation and plankton pro-duction The relative contribution of these sources dependson the region of the estuarine-lagoonal system and the depthof sediment In this sense stations of the north region (1 2and 3) are more submitted to OM from the Ribeira de IguapeRiver whereas samples 4 and 5 are submitted to both OMfrom the Ribeira de Iguape River and seawater with differentcontribution along the sedimentary cores Stations of thesouth region near Cananeia Island (9 10 11 and 12) are moresubmitted to OM from surrounding vegetation (mangrove)which dominates in all depths In addition stations locatedin the passage of seawater such as 6 and 8 are submitted toOM from surrounding vegetation (mangrove) and seawaterwhich presents more contribution in the first cm of thesedimentary cores

34 Contamination by Human Activities In addition toevaluating the profiles of sterols and triterpenols in the sedi-mentary core samples from the Cananeia-Iguape estuarine-lagoonal system we were also interested in assessing thelevels of contamination by sewage discharge in this envi-ronment Fecal sterols coprostanol and epicoprostanol weredetected in 30 and 10 among the 60 samples analyzedrespectively Coprostanol concentration ranged from 10 to

44 ngg whereas epicoprostanol ranged from 11 to 13 nggThe sedimentary core samples collected near to the Ribeira deIguape River were the ones with the highest concentrations ofcoprostanol which is a consequence of the input of domesticsewage from Iguape city that has a permanent population of30259 inhabitants The lowest concentrations of coprostanolfor sedimentary core samples collected near to Cananeia citymay be associated with its lower permanent population of12601 inhabitants

For comparative purposes concentrations of coprostanolobtained for the Cananeia-Iguape estuarine system werecompared to other estuarine and coastal environments inBrazilThe highest concentration of coprostanol found herein(44 ngg for station 1 and depth of 0ndash2 cm) is much lowerthan those found in the Babitonga Bay that has a permanentpopulation of 620000 inhabitants (4040 ngg) [3] in theinner shelf adjacent to Sergipe River that crosses Aracaju citywith a population of 2million inhabitants (184 ngg) [16] andin the Itajaı-Acu estuary (8930 ngg) that has a populationof 200000 inhabitants in Itajaı city and about 15 million inthe mesoregion [30] However the highest concentration ofcoprostanol found herein is similar to that found for Santosestuary that is near to a city with ca 420000 inhabitants(48 ngg) [15]

The concentration of 10 ngg is considered a thresholdvalue for sediments from natural coastal environments [14]In addition concentration higher than 500 ngg is consid-ered indicative of accentuated contamination by domesticsewage [14 30] The concentration levels of coprostanolobserved herein (10 to 44 ngg) are similar to or slightlyhigher than the threshold value (10 ngg) As there is noconsensus regarding the coprostanol concentration that isexclusively from domestic sewage diagnostic ratios are alsoused to enhance the reliability of the assessment of con-tamination [31 32] In this sense coprostanol(coprostanol+ cholestanol) and coprostanolcholesterol ratios were cal-culated to evaluate the level of sewage contamination inthe sedimentary core samples from Cananeia-Iguape sys-tem Based on these ratios sewage influence is not evidentfor the majority of sediment samples from the Cananeia-Iguape estuarine-lagoonal system (Figure 4) which pre-sented coprostanolcholesterol values that were lt02 andcoprostanol(coprostanol + cholestanol) values that werelt03 [31 32] The samples that fall in the region consideredcontaminated in one of the ratios correspond mainly tosedimentary core collected at station 1 which is submitted tothe OM coming from the Ribeira de Iguape River

Although some samples presented concentration ofcoprostanol higher than the threshold value and diagnosticratios that indicate contamination by domestic sewage tothe estuarine system these results suggest a moderate inputonce concentration of coprostanol higher than 500 ngg andcoprostanol(coprostanol + cholestanol) ratio higher than 07are found for environments submitted to accentuated input

4 Conclusions

In summary this is the first study that assessed steroland triterpenol biomarkers in sedimentary cores of the


00 02 04 06 08 10

Contaminated in both ratios

Coprostanolcholesterol

Uncontaminated in both ratios00

02

04

06

08

10

Cop

rost

anol

(cop

rost

anol

+ ch

oles

tano

l)

Figure 4 Plot of coprostanol(coprostanol + cholestanol) ratiosagainst coprostanolcholesterol ratios for all sedimentary core sam-ples that presented quantifiable concentration of these sterols Thelines inside the graph represent the regions that are consideredcontaminated or uncontaminated by the diagnostic ratios

Cananeia-Iguape estuarine-lagoonal system Typical dis-tribution of sterols and triterpenols was observed for eachregion of the study area The region near the Ribeira deIguape River is mainly submitted to OM of its freshwateroutflow presenting biomarkers of higher plants aquaticplants and plankton production As pointed by coprostanol(coprostanol + cholestanol) and coprostanolcholesterolratios and the absolute concentrations of coprostanol thisarea also presented amoderate sewage input as a consequenceof the proximity to Iguape cityThe south region is character-ized by a strong input of OM from surrounding vegetationmainly mangroves as revealed by the high proportions of120573-sitosterol and 120573-amyrin At the same time all regionssubmitted to seawater inflow present a higher contributionof cholesterol mainly in the first cm Therefore the assess-ment of sterols and triterpenols by UHPLC-MSMS gave usinsights into source transport and deposition of OM in theCananeia-Iguape estuarine-lagoonal system

Competing Interests

The authors declare that they have no competing interests

References

[1] E Benfenati E Cools E Fattore and R Fanelli ldquoA GCndashMSmethod for the analysis of fecal and plant sterols in sedimentsamplesrdquo Chemosphere vol 29 no 7 pp 1393ndash1405 1994

[2] E C Richard C Hamacher C O Farias et al ldquoHistoricalevolution of organicmatter accumulation in aCoastal Bay in theSW Atlantic Brazil use of sterols and n-Alcohols as molecularmarkersrdquo Journal of the Brazilian Chemical Society vol 25 no8 pp 1380ndash1390 2014

[3] C C Martins A C Cabral S C T Barbosa-Cintra A L LDauner and FM Souza ldquoAn integrated evaluation ofmolecularmarker indices and linear alkylbenzenes (LABs) to measure

sewage input in a subtropical estuary (Babitonga Bay Brazil)rdquoEnvironmental Pollution vol 188 pp 71ndash80 2014

[4] J I Hedges and R G Keil ldquoOrganic geochemical perspectiveson estuarine processes sorption reactions and consequencesrdquoMarine Chemistry vol 65 no 1-2 pp 55ndash65 1999

[5] S Bouillon and H T S Boschker ldquoBacterial carbon sourcesin coastal sediments a cross-system analysis based on stableisotope data of biomarkersrdquoBiogeosciences vol 3 no 2 pp 175ndash185 2006

[6] AC Spivak EACanuel J EDuffy and J P Richardson ldquoTop-down and bottom-up controls on sediment organic mattercomposition in an experimental seagrass ecosystemrdquo Limnologyand Oceanography vol 52 no 6 pp 2595ndash2607 2007

[7] E A Canuel A C Spivak E J Waterson and J E Duffy ldquoBio-diversity and food web structure influence short-term accumu-lation of sediment organic matter in an experimental seagrasssystemrdquo Limnology and Oceanography vol 52 no 2 pp 590ndash602 2007

[8] L Puerari R S Carreira A C B Neto L C Albarello and FD C Gallotta ldquoRegional assessment of sewage contaminationin sediments of the iguacu and the Barigui rivers (Curitiba CityParana Southern Brazil) using fecal steroidsrdquo Journal of theBrazilian Chemical Society vol 23 no 11 pp 2027ndash2034 2012

[9] S L McCallister J E Bauer H W Ducklow and E A CanuelldquoSources of estuarine dissolved and particulate organic mattera multi-tracer approachrdquo Organic Geochemistry vol 37 no 4pp 454ndash468 2006

[10] R A Moreau B D Whitaker and K B Hicks ldquoPhytosterolsphytostanols and their conjugates in foods structural diversityquantitative analysis and health-promoting usesrdquo Progress inLipid Research vol 41 no 6 pp 457ndash500 2002

[11] J K Volkman S M Barrett S I Blackburn M P Mansour EL Sikes and FGelin ldquoMicroalgal biomarkers a review of recentresearch developmentsrdquo Organic Geochemistry vol 29 no 5ndash7pp 1163ndash1179 1998

[12] P A Cranwell G Eglinton and N Robinson ldquoLipids of aquaticorganisms as potential contributors to lacustrine sediments-IIrdquoOrganic Geochemistry vol 11 no 6 pp 513ndash527 1987

[13] CHVane AWKim SMcGowan et al ldquoSedimentary recordsof sewage pollution using faecal markers in contrasting peri-urban shallow lakesrdquo Science of the Total Environment vol 409no 2 pp 345ndash356 2010

[14] I Tolosa M Mesa and C M Alonso-Hernandez ldquoSteroidmarkers to assess sewage and other sources of organic contam-inants in surface sediments of Cienfuegos Bay Cubardquo MarinePollution Bulletin vol 86 no 1-2 pp 84ndash90 2014

[15] G A Bataglion E Meurer A C R de Albergaria-Barbosa MC Bıcego R R Weber and M N Eberlin ldquoDetermination ofgeochemically important sterols and triterpenols in sedimentsusing ultrahigh-performance liquid chromatography tandemmass spectrometry (UHPLCminusMSMS)rdquo Analytical Chemistryvol 87 no 15 pp 7771ndash7778 2015

[16] R S Carreira A C Albergaria-BarbosaM L Arguelho and CA Garcia ldquoEvidence of sewage input to inner shelf sedimentsin the NE coast of Brazil obtained by molecular markersdistributionrdquoMarine Pollution Bulletin vol 90 no 1-2 pp 312ndash316 2015

[17] M A Goni M B Yunker R W Macdonald and T I EglintonldquoThe supply and preservation of ancient and modern compo-nents of organic carbon in the Canadian Beaufort Shelf of theArctic OceanrdquoMarine Chemistry vol 93 no 1 pp 53ndash73 2005


[18] J I Hedges and J M Oades ldquoComparative organic geo-chemistries of soils and marine sedimentsrdquo Organic Geochem-istry vol 27 no 7-8 pp 319ndash361 1997

[19] C Biache and R P Philp ldquoThe use of sterol distributionscombined with compound specific isotope analyses as a toolto identify the origin of fecal contamination in riversrdquo WaterResearch vol 47 no 3 pp 1201ndash1208 2013

[20] J Lembcke U Ceglarek GM Fiedler S Baumann A Leichtleand J Thiery ldquoRapid quantification of free and esterifiedphytosterols in human serum using APPI-LC-MSMSrdquo Journalof Lipid Research vol 46 no 1 pp 21ndash26 2005

[21] S Mo L Dong W J Hurst and R B van Breemen ldquoQuanti-tative analysis of phytosterols in edible oils using APCI liquidchromatography-tandem mass spectrometryrdquo Lipids vol 48no 9 pp 949ndash956 2013

[22] M M de Mahiques R C L Figueira A B Salaroli D P VAlves and C Goncalves ldquo150 years of anthropogenic metalinput in a Biosphere Reserve the case study of the Cananeia-Iguape coastal system Southeastern Brazilrdquo EnvironmentalEarth Sciences vol 68 no 4 pp 1073ndash1087 2013

[23] H H F Koolen E R Soares F M A da Silva A Q Lde Souza E R Filho and A D L de Souza ldquoTriterpenesand flavonoids from the roots of Mauritia flexuosardquo BrazilianJournal of Pharmacognosy vol 22 no 1 pp 189ndash192 2011

[24] P MMedeiros and B R T Simoneit ldquoMulti-biomarker charac-terization of sedimentary organic carbon in small rivers drain-ing the Northwestern United StatesrdquoOrganic Geochemistry vol39 no 1 pp 52ndash74 2008

[25] R A Andersson and P A Meyers ldquoEffect of climate changeon delivery and degradation of lipid biomarkers in a Holocenepeat sequence in the Eastern European Russian ArcticrdquoOrganicGeochemistry vol 53 no 1 pp 63ndash72 2012

[26] R S Carreira M P Araujo T L F Costa G Sporl and B AKnoppers ldquoLipids in the sedimentary record as markers of thesources and deposition of organic matter in a tropical Brazilianestuarine-lagoon systemrdquo Marine Chemistry vol 127 no 1ndash4pp 1ndash11 2011

[27] M A Ribeiro B A Knoppers and R S Carreira ldquoFontese distribuicao de materia organica sedimentar no com-plexo estuarino-lagunar de Mundau-ManguabaAL utilizandoesterois e alcoois como indicadoresrdquoQuımica Nova vol 34 no7 pp 1111ndash1118 2011

[28] S S OrsquoReilly M T Szpak P V Flanagan et al ldquoBiomarkersreveal the effects of hydrography on the sources and fate ofmarine and terrestrial organic matter in the western Irish SeardquoEstuarine Coastal and Shelf Science 2013

[29] R Jaffe R Mead M E Hernandez M C Peralba and O ADiGuida ldquoOrigin and transport of sedimentary organic matterin two subtropical estuaries a comparative biomarker-basedstudyrdquo Organic Geochemistry vol 32 no 4 pp 507ndash526 2001

[30] M Frena G A Bataglion A E Tonietto M N Eberlin M RAlexandre and L A Madureira ldquoAssessment of anthropogeniccontamination with sterol markers in surface sediments ofa tropical estuary (Itajaı-Acu Brazil)rdquo Science of The TotalEnvironment vol 544 no 1 pp 432ndash438 2016

[31] V Furtula H Osachoff G Derksen H Juahir A Colodey andP Chambers ldquoInorganic nitrogen sterols and bacterial sourcetracking as tools to characterize water quality and possiblecontamination sources in surface waterrdquo Water Research vol46 no 4 pp 1079ndash1092 2012

[32] T J Tse G Codling P D Jones et al ldquoReconstructing long-term trends in municipal sewage discharge into a small lake in

northern Manitoba Canadardquo Chemosphere vol 103 no 1 pp299ndash305 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


several algal classes whereas cholestanol often occurs in thedinoflagellates class [11] Other sterols such as dinosterol andergosterol are exclusive biomarkers of OM from diatoms andfungi respectively [12]

There are also important sterol biomarkers for anthro-pogenic OM which are known as fecal sterols (egcoprostanol and epicoprostanol) and indicate contaminationfrom domestic sewage [13] Some authors point that absoluteconcentration of coprostanol should not be used as a uniqueand conclusive parameter to assess sewage contaminationbecause of the possibility of in situ production To solve thisissue and to evaluate the degree of contamination by domesticsewage input some ratios such as coprostanol(coprostanol+ cholestanol) and coprostanolcholesterol have been pro-posed [14] Higher plants especially angiosperms displaytriterpenols as typical and exclusive biomarkers Triterpenolssuch as lupeol and 120572- and 120573-amyrin are the most commontriterpenols found in sediments of riverine and estuarineenvironments [15]

Estuarine systems are essential compartments involvedin the OM recycling and global carbon cycle [16] Under-standing the flows of OM frommarine and rivers to estuariesprovides information about its cycle in both water columnand sediments [17] Once OM in these systems constitutesa complex mixture of compounds from different sources areliable technique is required to overcome this complexity[18] Sterols in sediment samples are generally analyzed bygas chromatography-mass spectrometry (GC-MS) whichrequires a cleanup step using alumina and silica columnswhere sterols are separated from other classes of compoundsThe fraction containing sterols is usually derivatized toincrease their volatility and decrease the limits of detec-tion (LOD) [19] In this sense liquid chromatography (LC)as a separation technique and tandem mass spectrometry(MSMS) as a detection method are combined to over-come the inherent challenges of OM complexity [15] Somemethods employing analytical platforms based on liquidchromatograph tandem mass spectrometry (LC-MSMS) forthe analysis of selected sterols have been described over thelast years for a range of matrices [20 21] and more recentlyfor sediments [15]

Information on sources transport and deposition of OMis scarce for the Cananeia-Iguape estuarine-lagoonal systemwhich is an important Brazilian ecosystem from the WorldHeritage List under natural criteria (UNESCO) due to exten-sive areas of mangrove and Atlantic forest Consequentlythis study aimed to determine the sterol and triterpenolcomposition and assess the OM origin in sedimentary coresalong the study area Based on that an initial insight intothe geochemistry and flow of OM in this estuarine-lagoonalenvironment was achieved

2 Experimental

21 Study Area and Sampling The Cananeia-Iguape estua-rine-lagoonal system (Figure 1) is located in the SoutheastRegion of Brazil (Sao Paulo state) The opening of a 4 kmlong artificial channel linking the Ribeira de Iguape River tothe Cananeia-Iguape estuarine-lagoonal system in the XIX

Icapara outfall

Cananeacuteia outfall

Ribeira de Iguape River

Atlantic Ocean

S

N

W E

S1S2

S3

S4

S5S6S7

S8

S9S10

S11 S12

5 10 200(km)

Figure 1 Map of study area showing the 12 sampling stationsalong the Cananeia-Iguape estuarine-lagoonal system located inSao Paulo state (Southeast Brazil) Content may not reflect NationalGeographicrsquos current map policy Sources National GeographicEsri DeLorme HERE UNEP-WCMC USGS NASA ESA METINRCAN GEBCO NOAA and Increment P Corp

century caused an enormous impact in this environmentAfter its opening the channel eroded laterally reaching awidth and depth of approximately 250 and 7m respectivelyThus the channel was renamed to Valo Grande (ldquoBig Scourrdquo)in reference to the dimensions that it acquired As a result ofthis erosion environmental changes in the Cananeia-Iguapeestuarine-lagoonal system were intensified since about 60of the main flow of the Ribeira de Iguape River was trans-ferred to the system [22] The affected area is an importantecosystem from the World Heritage List located betweentwo cities (Cananeia and Iguape) with a protected greenarea consisting of typical vegetation of salt marshes Atlanticforest and mangroves [22]

A total of 60 samples from 12 collection sites (0ndash10 cmdepth) were collected at the Cananeia-Iguape estuarine-lagoonal system Sampling sites with their geographicallocations and lithological characteristics are shown in Table 1Sedimentary core samples were collected using a RossfelderVT-1 vibracorer and stored at 4∘C during transportation tothe laboratory Prior to the laboratory work all sedimentsamples were freeze-dried pulverized in a mortar and sievedwith a stainless steel sieve (lt250120583m) and stored at 4∘C beforeanalysis

22 Chemicals and Reagents (3120573)-Cholest-5-en-3-ol (cho-lesterol ge99 purity) (31205735120572)-cholestan-3-ol (cholestanolgt95 purity) (31205735120573)-cholestan-3-ol (coprostanol gt95purity) (31205725120573)-cholestan-3-ol (epicoprostanol gt95 pu-rity) (312057322E)-ergosta-5722-trien-3-ol (ergosterol gt95purity) (312057322E)-ergosta-522-dien-3-ol (brassicasterolgt95 purity) (312057324R)-ergost-5-en-3-ol (campesterol gt65purity) (3120573)-stigmast-5-en-3-ol (120573-sitosterol gt98 purity)(312057322E)-stigmasta-522-dien-3-ol (stigmasterol gt95 puri-ty) (31205735120572)-stigmastan-3-ol (stigmastanol gt95 purity)and (3120573)-lup-20(29)-en-3-ol (lupeol ge94 purity) were

















12

3

45 6

7

8910

110

250000500000750000

100000012500001500000

Inte

nsity








0

10

20

Rela

tive (

)

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

Relat

ive (

)

0

10

20

Rela

tive (

)

S1S2

S3

S4 S5 S6

S7 S8 S9

S10 S11 S12

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

40

50

60

Rela

tive (

)

2 3 4 5 6 7 8 9 10 11 12 131Compound

1 3 4 5 6 7 8 9 10 11 12 132Compound

1 3 4 5 6 7 8 9 10 11 12 132Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

40

Rela

tive (

)

0

10

20

30

40

50

Rela

tive (

)

0

10

20

30

40

Rela

tive (

)

010203040506070

Rela

tive (

)

0

10

20

30

Rela

tive (

)

0

10

20

30

Relat

ive (

)

0

10

20

30

40

50

Rela

tive (

)

0

10

20

30

40

50

Rela

tive (

)














4 Conclusions



00 02 04 06 08 10




02

04

06

08

10

Cop

rost

anol

(cop

rost

anol

+ ch

oles

tano

l)



Competing Interests


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

















12

3

45 6

7

8910

110

250000500000750000

100000012500001500000

Inte

nsity








0

10

20

Rela

tive (

)

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

Relat

ive (

)

0

10

20

Rela

tive (

)

S1S2

S3

S4 S5 S6

S7 S8 S9

S10 S11 S12

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

40

50

60

Rela

tive (

)

2 3 4 5 6 7 8 9 10 11 12 131Compound

1 3 4 5 6 7 8 9 10 11 12 132Compound

1 3 4 5 6 7 8 9 10 11 12 132Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

40

Rela

tive (

)

0

10

20

30

40

50

Rela

tive (

)

0

10

20

30

40

Rela

tive (

)

010203040506070

Rela

tive (

)

0

10

20

30

Rela

tive (

)

0

10

20

30

Relat

ive (

)

0

10

20

30

40

50

Rela

tive (

)

0

10

20

30

40

50

Rela

tive (

)














4 Conclusions



00 02 04 06 08 10




02

04

06

08

10

Cop

rost

anol

(cop

rost

anol

+ ch

oles

tano

l)



Competing Interests


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0

10

20

Rela

tive (

)

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

Relat

ive (

)

0

10

20

Rela

tive (

)

S1S2

S3

S4 S5 S6

S7 S8 S9

S10 S11 S12

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

40

50

60

Rela

tive (

)

2 3 4 5 6 7 8 9 10 11 12 131Compound

1 3 4 5 6 7 8 9 10 11 12 132Compound

1 3 4 5 6 7 8 9 10 11 12 132Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

2 3 4 5 6 7 8 9 10 11 12 131Compound

0

10

20

30

40

Rela

tive (

)

0

10

20

30

40

50

Rela

tive (

)

0

10

20

30

40

Rela

tive (

)

010203040506070

Rela

tive (

)

0

10

20

30

Rela

tive (

)

0

10

20

30

Relat

ive (

)

0

10

20

30

40

50

Rela

tive (

)

0

10

20

30

40

50

Rela

tive (

)














4 Conclusions



00 02 04 06 08 10




02

04

06

08

10

Cop

rost

anol

(cop

rost

anol

+ ch

oles

tano

l)



Competing Interests


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










4 Conclusions



00 02 04 06 08 10




02

04

06

08

10

Cop

rost

anol

(cop

rost

anol

+ ch

oles

tano

l)



Competing Interests


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


00 02 04 06 08 10




02

04

06

08

10

Cop

rost

anol

(cop

rost

anol

+ ch

oles

tano

l)



Competing Interests


References













































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

quantification of sterol and triterpenol biomarkers in sediments of

Documents